Cell, Vol. 13, 181-188,

January

1978,

Copyright

Isolation of Deletion Adenovirus Type 5

0 1978 by MIT

and Substitution

Nicholas Jones and Thomas Shenk Department of Microbiology University of Connecticut Health Center Farmington, Connecticut 06032

Summary The infectivity of adenovirus type 5 DNA can be increased to about 5 x lo3 plaque-forming units per pg DNA if the DNA is isolated as a DNAprotein complex. Utilizing this improved infectivity, a method was developed for the selection of mutants lacking restriction endonuclease cleavage sites. The procedure involves three steps. First, the DNA-protein complex is cleaved with a restriction endonuclease. The Eco RI restriction endonuclease was used here. It cleaves adenovirus type 5 DNA to produce three fragments: fragment A (O-O.76 map units), fragment C (0.760.83 map units) and fragment B (0.83-1.0 map units). Second, the mixture of fragments is rejoined by incubating with DNA ligase, and, third, the modified DNA is used to infect cells in a DNA plaque assay. Mutants were obtained which lacked the endonuclease cleavage site at 0.83 map units. Such mutant DNAs were selected by this procedure because they were cleaved by the Eco RI endonuclease to produce only two fragments: a normal A fragment and a fused B/C fragment. These two fragments could be rejoined to produce a viable DNA molecule as a result of a bimolecular reaction with one ligation event; this exerted a strong selection for such molecules since a trimolecular reaction (keeping the C fragment in its proper orientation) and two ligation events were required to regenerate a wild-type molecule. The alterations resulting in the loss of the Eco RI endonuclease cleavage site at 0.83 map units include both deletion and substitution mutations. The inserted sequences in the substitution mutations are cellular in origin. Introduction Conditional-lethal mutants of several adenovirus serotypes have been isolated and have proven to be valuable in the analysis of adenovirus genes (Williams et al., 1971; Takahashi, 1972; Ensinger and Ginsberg, 1972; Begin and Weber, 1975; Harrison, Graham and Williams, 1977). In general, the nucleotide sequence of these mutants probably differs from that of their wild-type parent by a single base pair change. Mutants with more substantial alterations of the viral DNA structure, such as deletion mutations, are also potentially useful in the genetic and biochemical analysis of the

Mutants

of

adenovirus genome. In fact, deletion mutants can offer several advantages not enjoyed by conditional lethal mutants. First, deletion mutants display a “nonleaky” phenotype-that is, the mutant protein is completely nonfunctional-and they do not revert spontaneously. Second, whereas conditionallethal mutations are generally restricted to those portions of genes which code for proteins, biochemically produced or selected deletion mutations can be introduced anywhere in the viral genome. This includes regions between genes, sequences which serve regulatory functions and regions within nonessential genes. The third advantage of deletion mutations is that they can be easily and precisely located on the physical map of the viral genome using bacterial restriction endonucleases and electron microscopic heteroduplex techniques. The very low infectivity of adenovirus DNA [307 oxw_t300

d,l303

\I~-l-sub304

I 80

/b.

I

1 b307 86

Figure 4. Portion of the Ad5 Physical Map (0.80-0.86 Showing the Alterations Present in the Mutant DNAs

Map Units)

The nature of the alterations was determined from the data in Figures 2 and 3 and Table 2. Ad5 segments which have been deleted are indicated by heavy bars, and insertions are designated by triangles. It is not known whether the mutation in several mutants is a very small deletion or a single base pair change. These mutations are marked by an “x.”

both a deletion and insertion; and mutants 306 and in 307 contain insertions with no evidence of deletions. The Inserted Sequences Are Cellular in Origin There were two possible origins of the inserted sequences present in some of the mutants: either they represent duplications of some segment of the Ad5 genome, or they are derived from the host cell. To distinguish between these possibilities, hybridization experiments were performed using strand-specific, 3ZP-labeled DNA probes prepared from restriction endonuclease-generated fragments of the mutant DNAs which contain the inserted sequences. DNAs prepared from the two mutants with the largest insertions, sub 305 and in 307, were cleaved with the Sma I endonuclease. The Sma I-B fragment (0.77-0.915 map units), which contained the inserted sequences, was isolated from each mutant. The normal Sma I-A fragment (0.195-0.38 map units) was also isolated from sub 305 to serve as a control in the hybridization experiments. The

20

40

60

DNAs Prepared Wild-Type and

The percentage of probe DNA appearing as double-stranded is plotted as a function of the molar ratio of unlabeled adenovirus DNA to probe DNA. (a) Probe DNA prepared from the Sma I-A fragment of sub 305 was reassociated with sob 305 DNA (0) or wt 300 DNA (0). (b) Probe DNA prepared from the Sma I-B fragment of sub 305 was reassociated with sub 305 DNA (0) of wt 300 DNA (0). (c) Probe DNA prepared from the Sma I-6 fragment of in 307 was reassociated with in 307 DNA (*) or wt 300 DNA ( 0).

fragments were labeled in vitro with 3ZP-dCTP using DNA polymerase I by the “nick translation” method of Rigby et al. (1977). Strand-specific probes were prepared from these labeled fragments by the complement excess procedure of Tibbeits and Pettersson (1974). To determine whether the insertions were duplications of Ad5 sequences, the 32P-labeled, singlestranded probe DNAs were hybridized to wild-type and the homologous mutant DNAs. If the insertions are Ad5-specific sequences, then the mutant probe DNA should hybridize to the same extent with a large excess of either mutant or wild-type DNA. If, however, the insertions are not viral sequences, a larger percentage of the mutant probe DNA will reassociate with mutant DNA than with wild-type DNA. As expected, the probe prepared from the Sma I-A fragment, which did not contain the insertion, hybridized to the same level with either sub 305 or wt 300 DNA (Figure 5a). The probe derived from the Sma I-B fragment of sub 305 DNA, however, reassociated with a great excess of wt 300 DNA to only 74% of the level to which it reassociated with homologous mutant DNA (Figure 5b); in 307 DNA reassociated with wt 300 DNA to 78% of the level to which it reassociated with homologous DNA (Figure 5~). The sequences in the mutant Sma I-B fragments which are not complementary to wildtype DNA (26% for sub 305 and 22% for in 307) must correspond to the inserted sequences. We conclude that the inserted sequences in sub 305 and in 307 DNAs are not derived from the Ad5 genome.

Cdl 166

In a similar experiment, the probe derived from the Sma I-B fragment of sub 305 reassociated with a large excess of in 307 DNA to only 74% of the level to which it reassociated with homologous DNA (data not shown). From this observation, we conclude that the insertions in sub 305 and in 307 DNAs are not identical. Consistent with this conclusion is the fact that the sub 305 insertion is cleaved by both the Hind III and Xho I restriction endonucleases, whereas the in 307 insertion is not cleaved by either enzyme (Figure 4). To obtain direct evidence that the insertions are derived from the host cell genome, the probes prepared from the mutant DNAs were reassociated with DNA prepared from uninfected HeLa cells using the procedure of Johansson et al. (1977). The single-stranded probe prepared from the Sma I-A fragment, which did not contain an insertion, did not reassociate at all with the cellular DNA (Figure 6). In contrast, significant levels of reassociation were obtained when probes derived from the mutant Sma I-B fragments were incubated with HeLa cell DNA. 4% of the sub 305 probe and 8.5% of the in 307 probe became double-stranded (Figure 6), indicating that the inserted sequences are cellular in origin. It is not clear why the observed plateau levels of reassociation are lower than predicted in both this (predicted 25%; obtained maximum of 8.5% in Figure 6) and the previous experiment (predicted 100%; obtained 75% in Figure 5a). This discrepancy is probably due to a technical problem and does not affect the basic conclusion drawn from the data-that the inserted sequences are derived from the host cell and not the Ad5 genome. Growth Characteristics of the Mutants All mutants described in this report are viable. The deletions and insertions present in these mutants are therefore situated in a region of the genome that is nonessential for vegetative growth. In Figure 7, the kinetics and yield of multiplication of some of the mutant viruses are compared to wt 300. All the mutants grow about as well as the wild-type. Isolation of Mutants Whose DNA Is Completely Resistant to Cleavage by the Eco RI Endonuclease No mutants lacking the Eco RI endonuclease cleavage site at 0.76 map units were isolated by the procedure described above. Mutants lacking this cleavage site were selected from sub 304 DNA. This mutant contains only one Eco RI endonuclease cleavage site-the site at 0.76 map units. This DNA was cleaved with the endonuclease, and the cleavage product was used to infect cells in a DNA plaque assay. DNA molecules containing the cleavage site at 0.76 map units would be cut into two fragments, while molecules lacking the site would

1 d2 2 n 8

s&t 305, Sma I-B

sub 305, Sml-A I I A I 2 3 4 5 MOLAR RATIO (CELL/PROBE) .t

.

Figure 6. Reassociation of 3ZP-Labeled Probe from Mutants sub 305 and in 307 with Unlabeled

DNAs Prepared HeLa Cell DNA

The hybridization reaction was run to a Cot of >104 mol-set/l (cell DNA concentration). The percentage of probe DNA appearing as double-stranded is plotted as a function of the molar ratio of unlabeled HeLa cell DNA to probe DNA. Probes were prepared from the Sma I-A fragment of sub 305 (A), the Sma I-B fragment of sub 305 (0) and the Sma I-B fragment of in 307 (0).

I

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I

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40 60 60 100 HOURS AFTER INFECTION

Figure 7. Growth Curves of Mutants in 293-31 Cells Cells were infected at a multiplicity of 5 pfu per cell. After adsorption for 60 min at 37”c, the monolayers were washed twice with Tris-buffered saline, and medium containing 10% fetal calf serum was added. Cultures were harvested at the indicated times, and the virus titer was measured by plaque assay on 29331 cells. (4) wt 300; (A) d/303; (0) sub 305; (0) in 307.

Ad5 Deletion 187

and Substitution

Mutants

remain intact and infectious. DNA derived from three of twelve clones tested in this experiment was completely resistant to cleavage by the Eco RI endonuclease (data not shown). The mobility of the Kpn I fragment F (0.725-0.81 on the Ad5 map), however, was not altered (data not shown) in any of the three mutant DNAs. The alterations of the Eco RI endonuclease cleavage site at 0.76 map units are therefore either very small deletions or single base pair changes. The growth kinetics and yield of these mutants were indistinguishable from wild-type virus (data not shown). Discussion The infectivity of Ad5 DNA can be increased about 1000 fold if the DNA is isolated as a DNA-protein complex. The end-associated protein described by Robinson et al. (1973) is possibly responsible for the increased infectivity observed for the complex, but we have no direct evidence as yet to support this supposition. Utilizing this improved infectivity, we have developed a biochemical procedure for the selection of viral DNAs which contain fewer restriction endonuclease cleavage sites than the general wild-type DNA population. Here we have selected Ad5 mutants which lack one of the two Eco RI endonuclease cleavage sites. We have also used this procedure to isolate Ad5 mutants lacking each of the four Xba I endonuclease cleavage sites (N. Jones and T. Shenk, unpublished data). This procedure therefore appears to be generally applicable, provided that the restriction endonuclease makes a limited number of cleavages in the DNA molecule (probably no more than four, given the present level of infectivity of Ad5 DNA). If the DNA is cut into too many pieces, the probability of reassembling the fragments in the proper order becomes too low, and infectious DNA molecules will not be produced at a detectable level. The mutants lacking the Eco RI endonuclease cleavage site at 0.835 map units contain deletions of as much as 2% of the Ad5 genome (0.83-0.85 map units in sub 304 and sub 305). Since these mutants are viable, this region is not required for lytic growth of Ad5. This region can also be deleted without affecting viability in the closely related Ad2 virus. Ad2+ ND,, a nondefective AD2-SV40 hybrid virus, lacks the Ad2 segment from 0.79-0.86 map units (Kelly and Lewis, 1973). This portion of the adenovirus genome codes for a 15,500 dalton polypeptide of unknown function which is expressed early after infection (Lewis et al., 1976). The nonessential region almost certainly does not extend all the way to the second Eco RI endonuclease cleavage site at 0.76 map units. Cleavage of Ad5 DNA with Eco RI endonuclease followed by treatment with DNA ligase would produce many

fused A/B molecules. If these molecules were viable, they would have been isolated in our experiment. Furthermore, all three mutants lacking the site at 0.76 map units contained very minor alterations (perhaps single base pair changes), suggesting that substantial alterations at this site cannot be tolerated if viability is to be maintained. Several of the mutants lacking the Eco RI endonuclease cleavage site at 0.835 map units contain insertions of host cell DNA in this region. Apparently this portion of the adenovirus genome acquires foreign DNA on occasion. This finding suggests that the nondefective adenovirus-SV40 hybrid viruses are a specific example of a more general phenomenon in adenoviruses. Here the foreign DNA acquired near 0.83 map units was a portion of the SV40 genome, and the hybrid was selected from the wild-type population, since it acquired the ability to grow more efficiently in monkey cells. Mutant in 307 contains an insertion of about 1700 base pairs, and no deletion of adenovirus sequences can be detected. Nevertheless, this mutant grows as well as wild-type virus, indicating that a viral DNA containing one or two additional genes can be packaged into infectious virions. Although the location in the Ad5 genome of one of the end points of the inserted cellular sequences is variable (about 0.835-0.85 map units), the other end point is always located between the Xho I and Eco RI endonuclease cleavage sites at 0.830 and 0.835 map units, respectively. Since the inserted sequences are different in at least two of the mutants (sub 305 and in 307), it is probable that several independent recombinational events have occurred in the region of 0.830-0.835 on the Ad5 genome. It is therefore possible that there is a site within the region at which recombinational exchanges between the viral and host genomes preferentially occur. Experimental

Procedures

Cells, Virus and Plaque Assays The adenovirus-transformed human embryo cell line (line 293-31) was provided by F. Graham and has been described (Harrison et al., 1977). The cells were maintained in Dulbecco’s modified minimal essential medium containing 10% fetal calf serum. Suspension cultures of HeLa 53 cells were grown in Eagle’s spinner medium containing 7% calf serum. The wild-type Ad5 (H5 wt 300) was a plaque-purified derivative of a virus stock originally received from H. Ginsberg. Plaque assays with either virus or DNA were performed on monolayers of 293-31 cells (Harrison et al., 1977). The (Ca),(POJ,precipitation method was used in infections with Ad5 DNA (Graham and Van der Eb, 1973). Enzymes The Eco RI restriction endonuclease was prepared and used according to published protocols (Morrow and Berg, 1972; Greene et al., 1973). The Kpn I restriction endonuclease was purchased from New England Biolabs, and the Sma I restriction endonuclease was a gift from C. Tibbetts. The reaction conditions

Cell 188

for these enzymes have been described: for Kpn I by Smith, Blattner and Davies, (1976), and for Sma I by Sambrook et al. (1975). DNA ligase from bacteriophage T4-infected E. coli B was obtained from Miles Laboratories. One unit of T4 DNA ligase converts 100 nmole of 3H-labeled poly [d(A.T.)] with an average chain length 100 nucleotides to an exonuclease Ill-resistant form in 30 min at 30°C (Modrich and Lehman, 1970). E. coli DNA polymerase I was purchased from Boehringer-Mannheim Biochemicals. DNAs Viral DNA was prepared from purified virions. Virus was purified as follows. Infected cells were suspended in a small volume of 0.1 M Tris-HCI (pH 8.0) and sonicated briefly at 4°C. Cellular debris was removed by centrifugation (3000 X g for 10 min), and the supernatant was layered on top of a two-step CsCl gradient (1.23 g/cc and 1.4 g/cc, 2 ml per step). After centrifugation (30,000 rpm for 2 hr in a Beckman SW41 rotor), the virus band was removed and further purified by centrifugation to equilibrium in CsCl (1.34 g/cc). DNA was extracted from the virions by the method of Pettersson and Sambrook (1973). Adenovirus DNAprotein complex was prepared by dissolving virions in guanidine hydrochloride (4 M) and centrifuging the complex to equilibrium in CsCl containing guanidine hydrochloride (4 M) (Robinson et al., 1973). Ad5 DNA-protein complex which was to be used as starting material for the selection of mutants was prepared from virions derived from a stock of wt 300 virus which had been serially passaged IO times without dilution through HeLa cells. The virus was serially passaged on the assumption that this treatment would favor the accumulation of variants containing altered genomes. HeLa cell DNA was prepared according to the method of Sharp, Petterson and Sambrook (1974). Gel Electrophoresis and Recovery of DNA from Agarose Gels DNA fragments produced by cleavage with restriction endonucleases were separated by electrophoresis in 0.7-l .O% agarose gels as described previously (Shenk, 1977). The method for extracting DNA fragments from agarose gels has also been described (Shenk, 1977). Preparation of Single-Stranded, Radioactively Labeled Probe DNAs Specific fragments of Ad5 DNA were prepared by cleavage with the Sma I restriction endonuclease, electrophoresis in 0.7% agarose gels and extraction of the appropriate fragments from the gels. The “nick translation” method of Rigby et al. (1977) was then used to introduce &*P-dCTP (113.9 Ci/mmole) into the fragments. The specific activity of the final product was 2-4 x 10’ cpm/pg, and ~5% of the radioactive product formed rapidly annealing structures after denaturation. The strands of the labeled DNA were separated by the complement-excess hybridization procedure of Tibbetts and Pettersson (1974) to produce a single-stranded probe DNA. The complementary strands of Ad5 DNA used in this procedure were prepared using poly(U,G) (Tibbetts et al., 1974). The poly(U.G) was a gift from C. Tibbetts. Hybridization Conditions Hybridization reactions were performed at 65°C in a solution containing sodium phosphate [O.l M (pH 6.8)], NaCl (1 M) and sodium dodecylsulphate (0.4%). Before hybridization, DNAs were sonicated to fragments 300-400 nucleotides in length. Duplex DNA was analyzed by hydroxylapatite chromatography (Sambrook, Sharp and Keller, 1972). Electron Microscopy Heteroduplex DNA was prepared for microscopy by the method of Davis, Simon and Davidson (1971) and examined in a Hitachi model 11 E electron microscope.

Acknowledgments We acknowledge the competent technical assistance of Ms. Bonnie Massey and Mrs. Bridget Prindle. This work was supported by a USPHS research grant from the National Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

September

29,1977;

revised

October

17,1977

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